Italian Institute for Nuclear Physics (INFN) partially funded this work through the INFN-MERIDIAN project. The authors acknowledge Prof. Edoardo Milotti (Physics Department, University of Trieste, Italy) for INFN-MERIDIAN project coordination; Dr. Roberto Chignola (Biotechnology Department, University of Verona, Italy) for providing the Caco-2 cells, the ohmmeter, and for his valuable help and training. We also acknowledge Agnese Solari for technical assistance.

The following protocol involves human blood withdrawal from healthy volunteers. Donors provided written informed consent prior to enrollment. This procedure is in accordance with the Helsinki Declaration and blood withdrawals were performed by a professional healthcare assistant.
1. Cell Culture and Co-culture Set-up
<ol>
	<li>One week before the irradiation, prepare a Caco-2 cell suspension containing 2.5 &#215; 105 cells/mL in fresh RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 &#956;g/mL streptomycin.</li>
	<li>Seed 2 mL of cell suspension in sterile 1 &#181;m-pore diameter cell culture inserts for 6-well plates and put the insert into a 6-well plate. 
	NOTE: Cell culture inserts might need to be activated by incubation with sterile complete medium prior to cell seeding. In this case, culture media should be discarded and replaced with cell suspension media.</li>
	<li>Add 3 mL of fresh RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 &#181;g/mL streptomycin in each bottom compartment, and culture the cells at 37 &#176;C in an incubator with humidified atmosphere containing 5% CO2.</li>
	<li>On the same day of Caco-2 cell irradiation, collect human whole blood in commercially available lithium-heparin coated 6 mL tubes (tube size: 13 x 100 mm).</li>
	<li>Subsequently, isolate peripheral blood mononuclear cells (PBMC) by using Ficoll gradient. To separate PBMC, put 25 mL of Ficoll in a 50 mL conical centrifuge tube and layer an equal volume of whole blood diluted 1:1 with RPMI1640 onto the Ficoll surface. 
	NOTE: A normal healthy donor usually has approximately 4 - 10 &#215; 106 PBMC/mL.</li>
	<li>Centrifuge the 50 mL tubes at 400 x g for 30 min at room temperature.</li>
	<li>Gently collect the PBMC at the interface between Ficoll and plasma by aspiration with a Pasteur pipette and put them in a 15 mL conical tube.</li>
	<li>Wash the PBMC twice by adding 10 mL of phosphate buffered saline (PBS) and centrifuging PBMC at 250 x g for 10 min.</li>
	<li>Culture PBMC for a maximum of 3 - 5 h in T25 cm2 flasks in complete RPMI1640 media, as described before, at 37 &#176;C in a humidified atmosphere containing 5% CO2. 
	NOTE: Collect PBMC on the day of the experiment and seed 2 &#215; 106 cells/well, suspended in 3 mL of complete RPMI1640 medium, in the bottom compartment of the co-culture. Inserts with Caco-2 cells are transferred in PBMC-containing wells 30 min after their irradiation.PBMC collected from whole blood cannot be maintained in culture for more than approximately 72 h, if not stimulated with e.g. phytohaemagglutinin (PHA), after which cells&#39; viability is lost. 
	CAUTION: The quality and safety of all blood and blood products must be guaranteed throughout the whole process.</li>
</ol>
2. Irradiation Set-up
Note: Irradiation of Caco-2 cells was performed at the radiotherapy department of Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) S. Maugeri (Pavia, Italy) with a linear accelerator routinely used to treat different types of cancers.
<ol>
	<li>Set photon X-rays energy to 6 MV peak. The linear accelerator operates in a pulsing regime (time between two consecutive pulses approximately of 4 ms; duration of a single pulse about 5 &#181;s with a dose rate up to 1 &#215; 10-3 Gy/p).</li>
	<li>Place the 6-well plates containing inserts with Caco-2 on the trajectory of X-rays on a 1.4 cm-thick Plexiglas sheet (corresponding to a distance slightly greater than the build-up of the used radiation) at 100 cm from the source of radiation.</li>
	<li>Place a 0.5 cm-thick bolus on each sample before the irradiation occurs to guarantee the backscattered radiation component and the charged particles equilibrium.</li>
	<li>Irradiate the cells (doses in the range 2 - 10 Gy) with a flat and symmetric (&#177; 2%) 20 x 20 cm2 radiation field and a dose-rate of 3 Gy/min. 
	NOTE: Control &#34;sham&#34;-irradiated cells underwent the same procedural conditions of the irradiated ones, without entering the irradiation room (received dose: 0 Gy). 
	CAUTION: Ionizing radiation-producing devices must be used only by specialized personnel.</li>
</ol>
3. Cell Viability Assay (MTT Assay)
Note: Caco-2 cell metabolic activity was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay14.
<ol>
	<li>Seed 2 &#215; 105 Caco-2 in a 24-well plate 24 h before irradiation in 1.25 mL of complete medium.</li>
	<li>Irradiate the cells as described in steps 2.1 - 2.4 then incubate the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>21 h after irradiation, add 100 &#181;L of 5 mg/mL MTT solution and keep the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for 3 h.</li>
	<li>Discard the supernatant and wash the cells with 1 mL of PBS.</li>
	<li>Dissolve formazan crystals by adding 500 &#181;L of dimethyl sulfoxide (DMSO) in each well and evaluate the absorbance with a multi-well plate reader at &#955; = 570 nm.</li>
	<li>Repeat step 3.3 - 3.4 - 3.5 - 3.6 at 45 h after irradiation. 
	NOTE: Results are shown as a perturbation from the corresponding sham condition, which is normalized to 100%. 
	CAUTION: DMSO is inflammable and irritating agent for skin, eyes and respiratory system (GHS07, GHS08). In case of contact with eyes, wash immediately with abundant water and seek medical advice. MTT is an irritating agent for skin, eyes and respiratory system (GHS07, GHS08). In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</li>
</ol>
4. Percentage of Viable Cells Determination (Trypan Blue Dye Exclusion Assay)
NOTE: Percentage of viable cells was assessed by Trypan Blue Dye Exclusion assay.
<ol>
	<li>Seed 2 &#215; 105 Caco-2 in 24-well plate 24 h before irradiation in 1.25 mL of complete medium.</li>
	<li>Irradiate the cells with doses in the range 0 - 10 Gy (see steps 2.1 - 2.4) then incubate the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for 24 - 48 h.</li>
	<li>After the two chosen time points, wash the cells with PBS, discard it, then add 100 &#181;L of Trypsin-ethylenediaminetetraacetic acid (EDTA) solution. Put the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for 2 min then add complete medium to arrest the enzymatic reaction.</li>
	<li>Collect the cells in a 1.5 mL-tube and centrifuge the tube at 500 x g for 5 min. Discard the supernatant and re-suspend the cells in 50 &#181;L of PBS.</li>
	<li>Mix the cell suspension with 50 &#181;L of Trypan Blue dye solution and incubate the mixture for 3 min at room temperature.</li>
	<li>Count the stained (non-viable) and unstained (viable) cells with a B&#252;rker chamber.</li>
</ol>
5. Trans-Epithelial Electrical Resistance (TEER)
<ol>
	<li>Seed 5 &#215; 105 Caco-2 cells one week prior to irradiation in 6-well plate co-culture inserts (polyethylene terephthalate (PET), 2 x 106 pores/cm2).</li>
	<li>1 h before irradiation, measure the TEER with a voltmeter/ohmmeter.</li>
	<li>Irradiate cells as described in steps 2.1 - 2.4.</li>
	<li>Incubate Caco-2 cells in presence/absence of co-culture with PBMC at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>For the first few hours post-irradiation, measure TEER every hour, and subsequently every 3 h up to 48 h.</li>
</ol>
6. Western Blot Analysis of Claudin-1, Occludin, Afadin, ZO-1, ZO-2, NF-kB, and XIAP
<ol>
	<li>Seed&#160;5 &#215; 105 cells 1 week before X-rays exposure in 6-well plate co-culture inserts (PET, 2 x 106 pores/cm2) and irradiate cells as described in steps 2.1 - 2.4.</li>
	<li>Incubate Caco-2 cells in presence/absence of co-culture with PBMC at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>48 h after irradiation, prepare Caco-2 and PBMC cell lysates by treating cells with cell lysis buffer and store samples at -20 &#176;C. 
	NOTE: the protocol can be paused here.</li>
	<li>Quantify total protein amount by bicinchoninic acid (BCA) method. 
	NOTE: the protocol can be paused here.</li>
	<li>Mix equal amounts of total proteins with Laemli Sample Buffer added with &#946;-mercaptoethanol (final concentration of &#946;-mercaptoethanol is 5%), heat the samples at 95 &#176;C for 5 min and spin them at 10,000 x g.</li>
	<li>Load samples in a 4 - 20% precast gel and perform the electrophoresis at 120 V for 1 h. Transfer proteins on a polyvinildiene fluoride (PVDF) membrane.</li>
	<li>Block non-specific binding sites by incubating PVDF membrane at room temperature with 5% non-fat dry milk (NFDM) in PBS added with 0.2% Tween-20 (PBT). Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.</li>
	<li>Incubate the membrane with gentle agitation for 1 h at room temperature prior to being placed overnight at 4 &#176;C with the following primary antibodies: anti-claudin-1, anti-ZO-1, anti-ZO-2, anti-afadin, anti-occludin, anti-NF-kB, anti-XIAP, and anti-actin. Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.</li>
	<li>Incubate the membranes with Horseradish Peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature with gentle agitation. Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.</li>
	<li>At room temperature, develop photographic films by incubating the membranes with enhanced chemo-luminescent kit.</li>
	<li>Acquire photographic films with a scanning system and quantify the obtained bands with suitable image analysis software. 
	NOTE: The evaluation of Claudin-1, Occludin, Afadin, ZO-1, and ZO-2 is performed in Caco-2 cells. The evaluation of NF-kB and XIAP is performed in PBMC. 
	CAUTION: &#946;-mercaptoethanol is toxic (GHS05, GHS06, GHS08, GHS09). Do not breathe vapors, avoid release into the environment, and wear individual protection devices and respiratory protection. If swallowed, immediately ask for medical advice.</li>
</ol>
7. Statistical analysis
<ol>
	<li>To determine whether the radiation exposure and the co-culture induce a statistically significant perturbation, perform a two-way analysis of variance (ANOVA) test with multiple comparisons for repeated measurements (with Bonferroni post-hoc tests to compare replicate means).</li>
	<li>If not otherwise stated, calculate the statistical significance (p) by two-tailed Student&#39;s t-test. Each value is the mean of &#8805;3 independent experiments &#177; Standard Error of the Mean (SEM).</li>
</ol>

<ol>
	<li>Cunningham, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer.">Colorectal cancer.</a> Lancet. 375 (9719), 1030-1047 (2010).</li><li>Mancuso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+bystander+radiation+effects+in+Patched+heterozygous+mouse+cerebellum.">Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum.</a> PNAS. 105 (34), 12445-12450 (2008).</li><li>Demaria, S., Formenti, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+abscopal+effects+of+local+radiotherapy+be+predicted+by+modeling+T+cell+trafficking.">Can abscopal effects of local radiotherapy be predicted by modeling T cell trafficking.</a> J Immunother Cancer. 4 (1), 29 (2016).</li><li>Trott, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+mechanisms+of+normal+tissue+damage:+Importance+for+the+design+of+NTCP+models.">Biological mechanisms of normal tissue damage: Importance for the design of NTCP models.</a> Radiother Oncol. 105 (1), 79-85 (2012).</li><li>Derer, A., Deloch, L., Rubner, Y., Fietkau, R., Frey, B., Gaipl, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio-immunotherapy-induced+immunogenic+cancer+cells+as+basis+for+induction+of+systemic+anti-tumor+immune+responses+-+pre-clinical+evidence+and+ongoing+clinical+applications.">Radio-immunotherapy-induced immunogenic cancer cells as basis for induction of systemic anti-tumor immune responses - pre-clinical evidence and ongoing clinical applications.</a> Front Immunol. 6, 505 (2015).</li><li>Frey, B., Gaipl, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio-immunotherapy:+The+focused+beam+expands.">Radio-immunotherapy: The focused beam expands.</a> Lancet Oncol. 16 (7), 742-743 (2015).</li><li>Prise, K. M., O'Sullivan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+bystander+signalling+in+cancer+therapy.">Radiation-induced bystander signalling in cancer therapy.</a> Nat Rev Cancer. 9 (5), 351-360 (2009).</li><li>Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., Zucco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier:+Influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics.">The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics.</a> Cell Biol Toxicol. 21 (1), 1-26 (2005).</li><li>Babini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+the+induction+of+apoptosis+mediated+by+radiation-induced+cytokine+release.">Mechanisms of the induction of apoptosis mediated by radiation-induced cytokine release.</a> Radiat Prot Dosim. 166 (1-4), 165-169 (2015).</li><li>Pellegrina, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+wheat+germ+agglutinin+on+human+gastrointestinal+epithelium:+Insights+from+an+experimental+model+of+immune/epithelial+cell+interaction.">Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell interaction.</a> Toxicol Appl Pharm. 237 (2), 146-153 (2009).</li><li>Pozo-Rubio, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunostimulatory+effect+of+faecal+Bifidobacterium+species+of+breast-fed+and+formula-fed+infants+in+a+peripheral+blood+mononuclear+cell/Caco-2+co-culture+system.">Immunostimulatory effect of faecal Bifidobacterium species of breast-fed and formula-fed infants in a peripheral blood mononuclear cell/Caco-2 co-culture system.</a> Brit J Nutr. 106 (8), 1216-1223 (2011).</li><li>Parlesak, A., Haller, D., Brinz, S., Baeuerlein, A., Bode, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+cytokine+release+by+differentiated+CACO-2+cells+in+a+compartmentalized+coculture+model+with+mononuclear+leucocytes+and+nonpathogenic+bacteria.">Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria.</a> Scand J Immunol. 60 (5), 477-485 (2004).</li><li>Haller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-pathogenic+bacteria+elicit+a+differential+cytokine+response+by+intestinal+epithelial+cell/leucocyte+co-cultures.">Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.</a> Gut. 47 (1), 79-87 (2000).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assay.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay.</a> J Immunol Methods. 65 (1-2), 55-63 (1983).</li><li>Morini, J., Babini, G., Barbieri, S., Baiocco, G., Ottolenghi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interplay+between+Radioresistant+Caco-2+Cells+and+the+Immune+System+Increases+Epithelial+Layer+Permeability+and+Alters+Signaling+Protein+Spectrum.">The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum.</a> Front Immunol. 8, 1-12 (2017).</li><li>Dittmar, S., Harms, H., Runkler, N., Maisner, A., Kim, K. S., Schneider-Schaulies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measles+virus-induced+block+of+transendothelial+migration+of+T+lymphocytes+and+infection-mediated+virus+spread+across+endothelial+cell+barriers.">Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers.</a> J Virol. 82 (22), 11273-11282 (2008).</li><li>Srinivasan, B., Kolli, A. R., Esch, M. B., Abaci, H. E., Shuler, M. L., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEER+Measurement+Techniques+for+In+Vitro+Barrier+Model+Systems.">TEER Measurement Techniques for In Vitro Barrier Model Systems.</a> J Lab Autom. 20 (2), 107-126 (2015).</li><li>Moyes, S. M., Killick, E. M., Morris, J. F., Kadhim, M. A., Hill, M. A., Carr, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+produced+by+external+radiation+in+parameters+influencing+intestinal+permeability+and+microparticle+uptake+in+vitro.">Changes produced by external radiation in parameters influencing intestinal permeability and microparticle uptake in vitro.</a> Int J Radiat Biol. 84 (6), 467-486 (2008).</li></ol>

The authors declare no conflict of interest.

Colorectal cancer, with its high occurrence in developed countries, is one of the most frequent causes of morbidity and mortality in the population. It is usually managed by surgery, chemotherapy, and radiotherapy1. In the framework of radiotherapy treatments, particular attention must be given to the effects of healthy tissue exposure4; moreover, systematic studies on the relationship between radiation exposure and the immune system are fundamental for developing approaches of radio-immunotherapy3.
The methodology we adopted in this work has been tailored to the investigation of Caco-2 cells and PBMC crosstalk. We focused on the effect of X-rays exposure of tumor cells, but the same protocol can be adapted to studies with pharmacological agents. Being closer to physiological conditions with respect to standard cell culturing, the strong advantage of this method is the possibility of a complete dissection of a complex system, given the possibility of analyzing both different cell types and the release of signal molecules into the two compartments of the co-culture itself. In this way, routinely applied biological methods can help the understanding of cellular interplay related mechanisms.
The initial characterization of X-ray-induced effects on Caco-2 cells was based on two complementary measurements, i.e. the MTT colorimetric assay and the Trypan blue dye exclusion test. The apparently inconsistent results could be explained by the different focus of these two assays. MTT assesses the oxidoreductase enzymes activity, while the Trypan blue dye is based on a living-cell exclusion dye mechanism.
The investigation of Caco-2-PBMC interplay requires the creation of an epithelial monolayer able to lead to a complete separation between the two compartments of the co-culture. The possibility of seeding Caco-2 cells in the co-culture insert allows the irradiation of only this cell population. Since the co-culture starts after irradiation, there is no bias due to any accidental exposure of PBMC. This set-up needs to be carefully handled to avoid damage (or contamination) to the cellular monolayer during the movements of the insert from one 6-well plate to the other. When carefully performed, TEER measurements are a simple and non-invasive method to investigate the monolayer permeability. This assay is strictly related to the co-culture set-up, and it is not the only choice for investigation of monolayer permeability. It allows a good reproducibility of measures once it is well calibrated with fresh complete medium. Common assays focus on the diffusion of chemical dyes from the upper &#34;apical&#34; compartment to the bottom &#34;basolateral&#34; one (e.g. fluorescein isothiocyanate (FITC)-dextran assay)16. However, since PBMC are present in the basolateral compartment in this study, we decided to avoid the introduction of chemical reagents in the experiments.
Among the different techniques adopted in this work, TEER measurement is the only one that requires the co-culture set-up17,18. However, the other common laboratory techniques provide more informative results when applied to cells in co-culture, as they allow the investigation of a setup closer to physiological conditions and the data have a stronger biological significance. On the other hand, it must be noted that the use of cells grown on a porous support could lead to some difficulties in the operations needed to prepare the samples, such as the cell lysis for the preparation of cellular extracts to be analyzed through western blotting.
The system adopted in this study has the potential to be further improved, e.g. with the application of substances able to recreate the extracellular matrix milieu. However, this will also result in an increased complexity of the system, and a complete dissection of the set-up will be more difficult to be achieved.
Setups for cell co-culture certainly represent a powerful tool for the advancement of in vitro research, and for the understanding of complex systems. This technique has the potential to increase the knowledge on fundamental mechanisms, providing new inputs to basic research studies on molecular signaling, and with possible applications to the modulation of the activity of the immune system in the framework of oncological clinical patient management.

The methodology adopted in this work was designed to investigate the interplay between colorectal cancer cells and the immune system, exploiting a set-up closer to the physiological condition when compared to normal 2-dimensional cell cultures.
Colorectal carcinoma (CRC) is considered the third most frequent type of cancer, with more than one million of cases worldwide (Global Cancer Observatory, International Agency for Research on Cancer, World Health Organization, http://gco.iarc.fr). Management of CRC is routinely performed through either surgery, chemotherapy or radiotherapy1. When compared to invasive techniques like surgery or chemotherapy, radiotherapy largely avoids the typical detrimental systemic reactions deriving from these clinical approaches, thanks to the localized delivery of radiation dose. However, side-effects can arise in the surrounding healthy tissues, triggering inflammation with direct damage to healthy cells and damage mediated by non-targeted effects2,3,4. Focusing on the adverse effects due to the irradiation during colorectal cancer treatment, two aspects need to be investigated. First, the mechanisms responsible of intestinal impermeability could be altered by radiation delivery causing, in turn, the possibility of side effects due to an altered containment of bacterial population and the paracellular passage of molecules and solutes. Second, the presence of gut-associated lymphatic tissue (GALT) acts as an outpost of the immune system, with the function of controlling bacterial growth and mediating the general immune response5,6,7. To fulfill these functions, intestinal impermeability is kept due to the function of junctional complexes between cells&#39; membranes. For these reasons, the induced detrimental consequences due to different doses of X-rays were investigated in Caco-2 cells alone and in co-cultures with PBMC.
Although conducting studies on cell cultures is the first setline of investigation in biomedical research, the lack of detailed knowledge of the mechanisms driving cell biology and reciprocal interactions between different cell types might become critical when approaching the study of physiology of organs, systems, and apparatuses that cannot be easily recreated in the laboratory. Therefore, we decided to adopt a co-culture set-up, allowing both the study of two cell populations together and the dissection of aspects related to intercellular and extracellular mechanisms.
Co-culture is a technique exploited when studying epithelial functions and the interplay between different cell types. In particular, the use of such a technique becomes mandatory in our case, because epithelia are made up of cells characterized by polarity. In the case of the intestinal barrier, enterocytes show a well-defined polarization, with apical and basolateral poles normally separated due to the presence of tight junction-creating adhesion molecules. This compartmentalization is needed for the tissue physiology, avoiding paracellular trafficking and allowing the passage of determined molecules only. This feature is of course impossible to recreate with a normal cell culturing set-up. Moreover, the adoption of the co-culture set-up reproduces the presence of immune cells only in the basolateral surface, while the apical surface (corresponding to intestinal lumen) is not directly in contact with other cells.
Recently, Caco-2 cell lines gained more importance as an in vitro model of intestinal barrier. Although derived from human colon adenocarcinoma, Caco-2 cells maintain the differentiation ability and create a functional polarized monolayer8, which allows the investigation of cell membrane properties when grown in a co-culture insert.
Since Caco-2 culturing on a porous membrane is a well-established in vitro model of intestinal monolayer, an improvement has been the co-culture between Caco-2 and other cells. This set-up has been adopted frequently to measure the crosstalk between different cell types9 and can be used to unravel Caco-2 perturbed response to exogenous stimuli when in co-culture, with respect to Caco-2 cultured alone.
Many studies have addressed Caco-2 behavior when co-cultured with both non-pathogenic bacteria and peripheral blood mononuclear cells, to elucidate in particular the crosstalk with the immune system10. Pozo-Rubio et al.11 studied the expression of several cytokines in a Caco-2/PBMC co-culture with bifidobacteria stimulating Caco-2 cells. Their work highlighted substantial modification to cytokine expression profiles depending on bacterial stimulation performed in presence/absence of PBMC. Their results lead to the conclusion that the presence of PBMC sensitizes Caco-2 to bifidobacteria.
Different responses of Caco-2 cells to non-pathogenic and pathogenic bacteria have been assessed by different research teams. Parlesak et al.12 demonstrated the immunosuppressive effects of Caco-2 cells on Escherichia coli-stimulated PBMC. Moreover, Haller et al.13 studied the response of Caco-2 cells stimulated with both lipopolysaccharide (LPS) from enteropathogenic E. coli spp. or non-enteropathogenic bacteria i.e.E. coli spp., Lactobacillus spp., strengthening the conjecture that the Caco-2 cells&#39; response strictly depends on the presence of leukocytes in the co-culture set-up.
By performing different complementary laboratory assays (e.g. western blot, trans-epithelial electrical resistance, MTT, etc.), in addition to the analysis of different cell types grown in co-culture, the whole methodology promises results that can be considered more representative of what really happens in vivo. Moreover, this set-up enables the separation of the different co-culture compartments, allowing not only the study of the cell types involved but also the intercellular signaling molecules released in the upper vs. lower compartment or in presence vs. absence of co-culture.

<table><tbody><tr><td>ThinCert&lt;br/&gt; 6 Well Cell Culture Inserts for Multiwell Plates</td><td>Greiner Bio-one</td><td>657610</td><td>Equipment</td></tr><tr><td>Cell Culture Multiwell Plate, 6 Well&amp;nbsp;</td><td>Greiner Bio-one</td><td>657160</td><td>Plastic</td></tr><tr><td>Cell Culture Multiwell Plate, 24 Well&amp;nbsp;</td><td>Greiner Bio-one</td><td>662160</td><td>Plastic</td></tr><tr><td>RPMI 1640 without L-Glutamine</td><td>Lonza</td><td>12-167F</td><td>Reagents</td></tr><tr><td>Foetal Bovine Serum</td><td>Lonza</td><td>DE14-801</td><td>Reagents</td></tr><tr><td>L-Glutamine 200 mM</td><td>Lonza</td><td>17-605C</td><td>Reagents</td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp; 10K/10K</td><td>Lonza</td><td>17-602E</td><td>Reagents</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Heal Force</td><td>HF240</td><td>Equipment</td></tr><tr><td>Ficoll Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td>Reagents</td></tr><tr><td>Tube, 50 mL, PP, Conical Bottom</td><td>Greiner Bio-one</td><td>227261</td><td>Plastic</td></tr><tr><td>Tube, 15 mL, PP, Conical Bottom</td><td>Greiner Bio-one</td><td>188261</td><td>Plastic</td></tr><tr><td>Centrifuge</td><td>ThermoScientific</td><td>CL31R</td><td>Equipment</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>P3813</td><td>Reagents</td></tr><tr><td>Cell Culture Flask, 25 cm&lt;sup&gt;2&lt;/sup&gt;, PS</td><td>Greiner Bio-one</td><td>690175</td><td>Plastic</td></tr><tr><td>Clinac 2100 Linear Accelerator</td><td>Varian</td><td>CLINAC 2100C/D</td><td>Equipment</td></tr><tr><td>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)</td><td>Sigma-Aldrich</td><td>M5655</td><td>Reagents</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td>Reagents</td></tr><tr><td>Multiwell Plate Reader</td><td>GDV</td><td>DV990BV6</td><td>Equipment</td></tr><tr><td>Trypan Blue Solution 0,4 %&amp;nbsp;</td><td>Amresco</td><td>K940</td><td>Reagents</td></tr><tr><td>Trypsin-EDTA Solution</td><td>Sigma-Aldrich</td><td>T4049</td><td>Reagents</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>0030125150</td><td>Reagents</td></tr><tr><td>Millicell-ERS voltmeter/ohmeter</td><td>Millipore</td><td>MERS 00001</td><td>Equipment</td></tr><tr><td>Cell Lysis Buffer (10x)</td><td>Cell Signalling Technology</td><td>#9803</td><td>Reagents</td></tr><tr><td>B&amp;uuml;rker Hemocytometer</td><td>Sigma-Aldrich</td><td>BR719520</td><td>Equipment</td></tr><tr><td>BCA Protein Quantification Kit</td><td>Abcam</td><td>ab102536</td><td>Reagents</td></tr><tr><td>2x Laemmli Sample Buffer&amp;nbsp;</td><td>BioRad</td><td>#1610737</td><td>Reagents</td></tr><tr><td>2-Mercaptoethanol&amp;nbsp;</td><td>BioRad</td><td>#1610710</td><td>Reagents</td></tr><tr><td>Accublock Digital Dry Bath</td><td>Labnet International Inc.</td><td>D1100</td><td>Equipment</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Stain-Free Protein Gels, 10 well, 30 &amp;micro;L</td><td>BioRad</td><td>#4568093&amp;nbsp;</td><td>Reagents</td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel</td><td>BioRad</td><td>#1658004</td><td>Equipment</td></tr><tr><td>PowerPac HC High-Current Power Supply</td><td>BioRad</td><td>#1645052&amp;nbsp;</td><td>Equipment</td></tr><tr><td>Precision Plus Protein WesternC Blotting Standards</td><td>BioRad</td><td>#1610385&amp;nbsp;</td><td>Reagents</td></tr><tr><td>10x Tris/Glycine/SDS Running Buffer</td><td>BioRad</td><td>#1610732&amp;nbsp;</td><td>Reagents</td></tr><tr><td>Trans-Blot&amp;nbsp;Turbo Mini PVDF Transfer Packs</td><td>BioRad</td><td>#1704156</td><td>Reagents</td></tr><tr><td>Trans-Blot&amp;nbsp;Turbo Transfer System</td><td>BioRad</td><td>#1704150</td><td>Equipment</td></tr><tr><td>Blotting-Grade Blocker&amp;nbsp;</td><td>BioRad</td><td>#1706404</td><td>Reagents</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>93773</td><td>Reagents</td></tr><tr><td>Claudin-1 (D5H1D) XP&amp;nbsp;Rabbit mAb&amp;nbsp;</td><td>Cell Signalling Technology</td><td>#13255</td><td>Reagents</td></tr><tr><td>Bovine Serum Albumine</td><td>Sigma-Aldrich</td><td>A7906</td><td>Reagents</td></tr><tr><td>ZO-1 (D7D12) Rabbit mAb&amp;nbsp;</td><td>Cell Signalling Technology</td><td>#8193</td><td>Reagents</td></tr><tr><td>ZO-2 Antibody&amp;nbsp;</td><td>Cell Signalling Technology</td><td>#2847</td><td>Reagents</td></tr><tr><td>Afadin (D1Y3Z) Rabbit mAb</td><td>Cell Signalling Technology</td><td>&amp;nbsp;#13531</td><td>Reagents</td></tr><tr><td>Anti-Occludin Antibody</td><td>Millipore</td><td>ABT146</td><td>Reagents</td></tr><tr><td>Anti-NF-kB p65 antibody [E379]</td><td>Abcam</td><td>ab32536</td><td>Reagents</td></tr><tr><td>Anti-XIAP antibody&amp;nbsp;</td><td>Abcam</td><td>ab21278</td><td>Reagents</td></tr><tr><td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td><td>GE Healthcare Life Sciences</td><td>NA931V</td><td>Reagents</td></tr><tr><td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td><td>GE Healthcare Life Sciences</td><td>NA934V</td><td>Reagents</td></tr><tr><td>Clarity Western ECL Substrate, 200 mL</td><td>BioRad</td><td>#1705060&amp;nbsp;</td><td>Reagents</td></tr><tr><td>Carestream&amp;nbsp;Kodak&amp;nbsp;autoradiography GBX developer/replenisher</td><td>Sigma-Aldrich</td><td>P7042</td><td>Reagents</td></tr><tr><td>Carestream&amp;nbsp;Kodak&amp;nbsp;autoradiography GBX fixer/replenisher</td><td>Sigma-Aldrich</td><td>P7167</td><td>Reagents</td></tr><tr><td>Carestream&amp;nbsp;Kodak&amp;nbsp;BioMax&amp;nbsp;light film</td><td>Sigma-Aldrich</td><td>Z370401</td><td>Reagents</td></tr><tr><td>Gel Doc EZ System</td><td>BioRad</td><td>#1708270&amp;nbsp;</td><td>Equipment</td></tr></tbody></table>

a co-culture method to investigate the crosstalk between x-ray irradiated caco-2 cells and pbmc

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

One week prior to the experiment, cells are sown onto the porous membrane of the insert and allowed to grow during the following days. The level of the confluence can be checked either using an inverted microscope or via the measurement of the TEER values. Indeed, during the growth phase, TEER keeps increasing until all the porous membrane has been covered by cells and they form a differentiated cell monolayer. If cells proliferate faster/slower, the experiment could start at earlier/later time points after being seeded. When at confluence, cells are then brought to the irradiation facility, minimizing the imparted environmental stress (temperature or pH), prior to starting the co-culture with/without PBMC, seeded in the bottom compartment (Figure 1A), or evaluating the proliferation of Caco-2 cells. Given the initial seeding density, on day 0 cells should reach 100% confluence and create a differentiated monolayer of epithelial cells, which can be observed by the plateau in TEER shown in Figure 1B. Once the cells reach such status, the TEER value is kept relatively constant over the following week, as long as the old culture medium is replaced with fresh medium, at least once a week (Figure 1B). As shown in Figure 1C, the MTT assay does not show any statistically significant alteration of the proliferative status of Caco-2 cells neither at 24 h nor at 48 h, independently of the dose received (up to 10 Gy).
A different result was observed concerning the short-term mortality of Caco-2 cells. At both time points, Trypan blue staining show a dose-dependent increase in cell mortality. These results show a clear effect of the radiation exposure, although the percentages of dead cells appear to be very low, particularly when considering that the highest delivered dose (10 Gy) produces only roughly 20% of cell death (Figure 1D).
Samples were co-cultured with or without PBMC in the lower compartment. Given the fact that PBMC did not receive any external stimulus to proliferate, a 48 h experiment was considered ideal to avoid the bias introduced by PBMC starting to die. Therefore, from immediately before the irradiation, TEER was regularly measured for 48 h, to keep track of possible transitory effects caused by the irradiation protocol. As shown in Figure 1 E-F, TEER values are presented as relative variations with respect to the pre-treated condition (which were of the order of 1200 - 1500 &#937;&#183;cm2) to better highlight the perturbation induced by the X-ray irradiation and/or by the presence/absence of PBMC in the co-culture. In both cases, an initial transient peak can be clearly seen at the first time point after the radiation exposure, which can be attributed to the irradiation procedure.
When not in co-culture with PBMC (Figure 1E), TEER values are almost constant up to 48 h while, after 10 Gy of X-rays, cells show a prolonged decrease in TEER beginning at 3 h post-irradiation. The presence of PBMCs completely modifies the TEER temporal dynamics (Figure 1F). For all doses, a reduction in the TEER is clearly observable from 3 h up to approximately 30 h post-irradiation, when TEER appears to settle at a constant value (Figure 1F).
Tight junction complexes expression levels were investigated in Caco-2 cells lysates through western blot assay. Caco-2 cells were exposed to ionizing radiation (with doses of 0, 2, and 10 Gy) and subsequently grown alone or in co-culture with PBMCs in the bottom compartment for 48 h (as shown in Figure 2A-F). Both Claudin-1 and Occludin (Figure 2A, 2B) were found to be not significantly altered by X-ray and/or co-culture with PBMC. Large fluctuations were instead observed in scaffold proteins ZO-1, ZO-2 and Afadin (Figure 2C, 2D, 2E). In particular, a reduction in ZO-2 expression levels is observed already after 2 Gy when in co-culture with PBMC while only at 10 Gy when Caco-2 were growing alone. Afadin expression levels instead are affected only after 10 Gy of X-rays, with an additional reduction when Caco-2 are co-cultured with PBMCs.
PBMCs co-cultured with Caco-2 were analyzed regarding the inflammatory state, in particular, the Nuclear Transcription Factor kB (NF-kB) and the X-linked inhibitor of apoptosis protein (XIAP) levels have been investigated (Figure 2 G-I). NF-kB total amount was not affected by the co-culture with Caco-2 exposed to different radiation doses (Figure 2G). On the contrary, XIAP levels were 4-fold up-regulated in both the 2 Gy and 10 Gy co-cultures, although the large variations demand a higher number of samples analyzed to reduce such fluctuations and gain a better statistical power.
As shown in Figure 2F and 2I, some non-specific bands might appear next to the expected molecular weight of the protein of interest. Unless true and non-specific bands are easily distinguishable, different antibody and/or BSA or NFDM concentrations should be considered.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56908/56908fig1.jpg" /> 
Figure 1. Overall experimental setup and macroscopic effects of radiation exposure and/or PBMC co-culture. A) Schematic depiction of the co-culture model. B) TEER values measured daily from the initial seed of Caco-2 cells to assess the proper growth and differentiation status of the monolayer. C) Cell viability and D) mortality in Caco-2 exposed to X-rays (0, 2, 5, and 10 Gy). E) TEER measurements in Caco-2 cells irradiated with 0, 2, and 10 Gy of X-rays cultured without or F) with PBMCs. Each value is the mean of &#8805;3 independent experiments &#177; SEM. * p-val &#60;0.05; ** p-va l&#60; 0.01; *** p-val &#60; 0.001. Graphs adapted from Morini et al.15. <a href="https://cloudflare.jove.com/files/ftp_upload/56908/56908fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56908/56908fig2.jpg" /> 
Figure 2.&#160;Western Blot results of Caco-2 and PBMC lysates. Expression level of the tight junction proteins (Claudin-1 (A), Occludin (B), ZO-1 (C), ZO-2 (D), and Afadin (E)) in Caco-2 after 0, 2, and 10 Gy of X-rays and in presence/absence of PBMC in co-culture. Values are normalized on Actin level. Illustrative films for each tight junction protein and conditions are shown in panel (F). Expression level of NF-&#954;B (G) and XIAP (H) in PBMCs co-cultured with Caco-2 cells. Representative films for NF-kB, XIAP, and Actin are shown in panel. Each value is the mean of &#8805;3 independent experiments &#177; SEM. Graphs adapted from Morini et al.15. <a href="https://cloudflare.jove.com/files/ftp_upload/56908/56908fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC at JoVE.com

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

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JoVE Journal

Immunology and Infection

13.0K Views.  Università degli Studi di Pavia. We presenteren een protocol om te onderzoeken de Overspraak tussen Caco-2 X-Vleug-bestraalde en perifere bloed mononucleaire cellen (PBMC). Het protocol start met Caco-2 bestraling en opzet van de co cultuur met PBMC; trans-epitheliale elektrische weerstand is vervolgens regelmatig gedurende 48 h en westelijke vlek uitgevoerd in zowel Caco-2 en PBMC gemeten.

Video: A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Co-culturing BC Cells: Culturing Breast Cancer Cells with Bone Fragments to Study Cancer Cell Proliferation

Source: Templeton, Z. S., et al. <a href="https://www.jove.com/v/52656/methods-for-culturing-human-femur-tissue-explants-to-study-breast">Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche.</a> J. Vis. Exp (2015).
This video describes the culturing of breast cancer cells adjacent to femur bone explants to measure cancer cell proliferation and colonization in a human bone tissue explant model system.

Source: Templeton, Z. S., et al. Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche. J. ...

JoVE Encyclopedia of Experiments

Breast Cancer

In vitro studies

3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

Source: Espinoza-S&#225;nchez, et al. <a href="https://www.jove.com/v/56589/analyzing-communication-between-monocytes-primary-breast-cancer-cells">Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System.</a> J. Vis. Exp. (2018).
This video describes the 3D co-culture of breast cancer cells with monocytes to study their interaction in an extracellular matrix extract (ECME) based environment. This method helps in studying specific features such as collagen degradation, immune cell recruitment, and cell invasion.

Source: Espinoza-S&#225;nchez, et al. Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

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